A structured illumination microscopy module using two micro-electromechanical system scanning micromirrors

Super-resolution microscopy techniques have seen significant advancements in recent decades, with approaches covering optical interference, selective fluorescence quenching, statistical blinking events or computational enhancements through artificial intelligence having been implemented. The technique most extensively used in live-cell imaging is structured illumination microscopy (SIM), which, through optical interference, achieves theoretical spatial resolution doubling in all dimensions without the stringent requirements on sample sparsity, morphology or special labels typical of other methods. The optical interference required for 2D SIM is generally obtained either by placing a fixed diffraction grating, or more recently, a configurable digital micromirror device (DMD) or spatial light modulator, in the excitation path and focusing the 1st orders onto the back aperture of a microscope objective thereby generating high resolution stripe patterns within the fluorescently labelled specimen.
We present a novel SIM implementation using two micro-electro-mechanical system (MEMS) micromirrors to allow flexible control of the excitation illumination with reduced spatial footprint and cost-efficient integration. This allows for direct implementation of multi-color imaging in a compact and adaptable package. The 2D SIM approach is enabled by two three-axis micromirrors with static positioning and piston control, which allow precise angular, radial and phase positioning of two interference beams in the back-aperture of a microscope objective. This way isotropic 2D resolution enhancement can be achieved while simultaneously allowing spatial frequency control of the interference pattern to circumvent the missing cone problem in static 2D SIM. The imaging performance and flexibility will be benchmarked against a system using a DMD to obtain 2D SIM.